Quantifying Cardiac Fibrosis and Microvasculature

The heart was perfused with heparinized PBS (1,000 U/500 mL) and fixed overnight with 4% paraformaldehyde (PFA, #163-20145, Fujifilm. Co. Ltd.), embedded in paraffin, and cut into 3- to 4-mm-thick sections. The sections were stained with picrosirius red (Muto Co., Ltd. Japan) to determine the size of interstitial fibrosis, as described previously (11 (link)). The immunohistochemistry data were evaluated from 3 to 5 high-power fields per section in a blinded manner using Image J (version 1.51, National Institutes of Health). The sections were blocked in an avidin and streptavidin complex and incubated with isolectin B4-FITC (1:100) (Invitrogen, CA, USA) to evaluate the infarcted tissue microvascular density (MVD) (12 (link)). To discriminate auto-fluorescence, spectral analysis of stained tissue sections was performed using a Carl Zeiss LSM880 meta confocal microscope (Oberkochen, Germany).